RNA localization, a ubiquitous cellular strategy for regulating the subcellular site of mRNA translation, operates via a common, staged mechanism. First, a cis-encoded localization sequence (?zipcode?) is recognized by RNA-binding proteins and the mRNA assembled into a translationally-silenced RNP transport complex. The RNP complex is then localized to the appropriate subcellular destination, either by diffusion or by active transport, and anchored. Lastly, translation of the mRNA is derepressed and local protein synthesis ensues. Although substantial progress has been made in identifying zipcode signals, trans-acting RNA binding proteins, molecular motors and transport mechanisms, very little is known regarding molecular mechanisms of mRNA anchoring, which is critical to the maintence of localized protein synthesis. In our research into mechanisms of mRNA localization and anchoring on the endoplasmic reticulum (ER), we discovered that organelle protein-encoding mRNAs are directly anchored to the ER membrane. In contrast, secretory protein-encoding mRNAs, which also localize to the ER, and are anchored indirectly, via translation on ER-bound ribosomes. We hypothesize that a direct RNA anchoring mechanism acts to spatially coordinate the synthesis of functionally related genes. To identify the mechanism of direct mRNA anchoring to the ER, we performed proteomic interactor screens of ER-bound polyribosomes and identified candidate ER integral membrane RNA anchoring proteins. In a first aim, functional validation studies of candidate RNA anchoring proteins will be performed. mRNA identities, cis-ER anchoring motifs, and RNA binding domains for candidate interactors will be identified via photocrosslinking and immunoprecipitation/RNA- Seq (CLIP-Seq) and PAR-CLIP approaches. Candidate ER-RNA anchoring protein function will be further validated through assays of target mRNA translation and localization, using siRNA knockdown and where available, knockout animal models, to determine roles for direct ER-mRNA anchoring in gene expression. The finding that mRNAs can be directly anchored to the ER suggests a novel mechanism of ribosome trafficking to the ER, where membrane-anchored mRNAs directly recruit ribosomes for de novo translation. In support of this model, we reported previously that ER-bound ribosomes function in de novo translation initiation and remain ER-associated following translation termination. Extending from these observations, we hypothesize that translation on the ER is functionally compartmentalized from cytosolic translation. A primary prediction of this model is that the ER translation cycle operates without an obligatory exchange of ribosomal subunits with a cytosolic pool. We propose to test this hypothesis in a second aim, where we will determine the subcellular site(s) of de novo translation initiation and the role of translation in the regulation of ribosome exchange on the ER. We expect that the proposed research will reveal new paradigms for the subcellular organization of mRNA translation and its regulation in health and disease.